miR-4780 Derived from N2-Like Neutrophil Exosome Aggravates Epithelial-Mesenchymal Transition and Angiogenesis in Colorectal Cancer

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Introduction
Colorectal cancer (CRC) is one of the most prevalent tumors worldwide [1]. Although considerable progresses have been made in diagnosing approaches and therapeutic strategies for CRC, the prognosis for patients with advanced stage is still poor due to the metastases, and usually distant metastasis contributes to high mortality [2]. Therefore, it is crucial to investigate the occurrence and development of metastases in CRC. Increasing evidence has revealed that tumor microenvironment (TME) also participates in cancer pathogenesis besides genetic alterations and dysregulation of intracellular pathways [3][4][5]. TME is associated with tumor generation, development, and metastasis [6,7]. It mainly contains various immune cells and tumor-related stromal fibroblasts, among which immune cells are one of the main components in the TME [8]. During the carcinogenesis, the change in microenvironment toward an inflammatory milieu and contributes to tissue remodeling and tumor progression; the dynamic tumor interaction with TME is therefore associated with the regulation of neoangiogenesis, the supply of growth factors and cytokines, and remodeling of the extracellular matrix (ECM), as well as immune escape and drug resistance [9][10][11][12]. It has become evident that stromal cells also contribute to the polarization and activation of immune cells within TME [13][14][15].

Neutrophil Polarization and Verification.
Human myeloid leukemia cell line NB-4 cells were purchased from ATCC (Bioleaf, China) and induced neutrophil polarization according to the previous manuscript [34]. In brief, 100 mg all-trans retinoic acid (ATRA, R2625, Sigma, MO, USA) powder was dissolved in 10 mL dimethyl sulfoxide under aseptic conditions to prepare a storage solution at a concentration of 33.2 mM/L. NB-4 cell line was cultured in RPMI-1640 culture medium containing 10% FBS in the presence of 5% CO 2 at 37°C. About 1 μmol/L ATRA was utilized to differentiate NB-4 to neutrophil-like cells for 4 or 5 days. The morphology of differentiated neutrophil-like NB-4 cells was observed under an optical microscope and verified by Wright's stain. The differentiated neutrophil-like NB-4 cells were separated into two groups, N0-like neutrophil (differentiated neutrophil-like NB-4 cells were left untreated) and N2-like neutrophil. In N2-like neutrophil group, neutrophillike NB-4 cells were induced to N2-like polarization by TGF-β under selected concentration for 24 hr according to different polarity disposal conditions of neutrophils [25]. miR-4780 inhibitor and its corresponding nonspecific vector were synthesized (Ribo Bio, China) and transfected into neutrophils, according to the manuscript.

Detection of Neutrophil Activation.
To screen the concentration of TGF-β on N2-like polarization, the proportion of CD11b and CD66b was detected by flow cytometer. ATRA-treated neutrophil-like NB-4 cells were collected, and TGF-β in different concentrations was applied. The 5 μL antibody to CD11b (ab24874, Abcam, UK) and CD66b (ab275587, Abcam, UK) was added, and cell fluorescence was detected under flow cytometry.
2.6. The Extraction and Uptake of Neutrophil-Derived Exosomes. Exosome isolation was performed via Exosome extraction reagent in the ratio of 1 : 4 (Ribo, China). About 300 mL supernatants of neutrophils were centrifuged at 2,000 g for 30 min to remove the precipitation. Then the supernatants containing exosomes were first concentrated using a 100 kDa ultrafiltration Vivaflow 200 module to 10-15 mL and to a final volume of between 0.5 and 1 mL by a 100 kDa ultracentrifuge tube (3,000 g × 30 min). Next, exosomes were precipitated by adding exosome extraction reagent at the ratio of 1 : 4 and incubated overnight at 4°C. Then the exosome precipitation was obtained by centrifugation at 1,500 g for 30 min, resuspended in phosphate-buffered saline (PBS). Exosome protein content was measured using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, CN). The isolated exosomes were observed and identified by transmission electron microscopy (TEM) [35].
About 60 μg of isolated exosomes were incubated with 1 mL PKH-67 green fluorescent dye for 5 min (1 × 10-3 mM, Sigma Aldrich, Germany) and washed to remove excess dye using 100 kDa filter. Then, the labeled exosomes were incubated with the recipient, namely, CRC cell lines COLO205 and SW480, respectively, for 48 hr, and then the recipients were stained with DAPI and photographed using a laserscanning confocal microscope. For flow cytometry, cell was fixed with precooled 70% alcohol for 24 hr, washed with PBS, and then incubated with propidium iodide (PI) (Dongren Chemical Technology Co., Ltd, Shanghai, China) under dark conditions at 4°C. Cell cycle stages were detected by a flow cytometer (Partec GmbH, CyFlow Space). Cell apoptosis was detected using the Annexin V-FITC/PI staining. 100 μL of 1 × 10 6 cells/mL cell suspension was added into 5 mL of Annexin V-FITC (Partec GmbH, CyFlow Space) and 10 μL of PI, and the culture was incubated at room temperature for 15 min and washed twice with PBS. Cell apoptosis was analyzed using a flow cytometer at an excitation wavelength of 488 nm.

Cell Proliferation and
2.8. Migration and Invasion Assay. Cell wound healing assay was used to detect the ability of cell migration. When cells were 80%-90% confluent, the cell monolayer was scraped with a sterile 200 μL pipette tip. After being incubated for 24 hr, the cells were fixed, and images were taken. The migration distance and mobility were calculated using ImageJ (version 1.80, National Institutes of Health, Bethesda, MD, USA).
For cell invasion assay, cell suspension (1 × 10 4 cells) in FBS-free medium was plated in the upper chamber coated with Matrigel (Corning, Costar, Corning, NY, USA) and incubated for 24 hr. After incubation, cells that had invaded the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet solution (Beyotime Biotechnology, China). The number of cell invasion in five random fields were counted using ImageJ.
2.9. Tube Formation. Human umbilical vein endothelial cells (HUVECs, ATCC, VA, USA, cat. No. PCS-100-010) were cultured with CRC cell lines in a ratio of 3 : 1 [36]. Matrigel was diluted to 5 mg/mL with serum-free Dulbecco's Modified Eagle Medium and plated at 37°C for 30 min to harden the Matrigel. Then, 1.5 × 10 4 cells were inoculated in 96-well plates on the pre-coated Matrigel for 6 hr at 37°C. Four random fields were taken at capillary-like structures were counted with ImageJ.
2.10. Quantitative Polymerase Chain Reaction (qPCR). TRIzol reagent (Invitrogen, USA) was used to extract the total RNA from exosomes or CRC cell lines COLO205 and SW480. Nanodrop ND-1000 (Nanodrop, DE, USA) was employed to verify the quality and quantity of extracted RNA. The integrity of RNA was assessed by standard denaturing agarose gel electrophoresis. PCR was performed using cDNA and SYBR Green Real-Time PCR Master Mixes (Thermo Fisher Scientific, USA) with initial denaturation at 95°C for 1 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 2 min, and extension for 30 s, at 72°C. Primers were designed and validated by Ribo Co. Ltd. Results of the log-linear phase of the growth curve were analyzed, and relative quantification was performed using the 2 −ΔΔCt method with β-actin and GAPDH as a housekeeping gene. Each experiment was repeated independently three times.
2.11. Western Blot. Cells and liver tissues from liver metastasis model were collected and lysed in radio immunoprecipitation assay lysis buffer (containing 0.01% phenylmethanesulfonyl fluoride, 150 nmol/L Tris (pH = 8), 0.1% SDS, 0.2% ethylenediaminetetraacetic acid, 1% Triton X-100, 1% sodium deoxycholate). BCA protein assay kit (KeyGen Biotech, China) was used to detect the protein concentration. The total protein samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked Stem Cells International using 5% skimmed milk at room temperature for 2 hr and then were probed with primary antibodies overnight at 4°C. Primary antibodies are listed in Table 1. After washing by TBS-T, membranes were incubated with horseradish peroxidaseconjugated secondary antibodies for 2 hr at room temperature. Enhanced chemiluminescence assay was used and captured through Imager. The optical density of the bands was analyzed using NIH ImageJ software.
2.12. Enzyme-Linked Immunosorbent Assay (ELISA). The concentrations of CXCL10, IL-12, IL-10, and CCL2 were measured via ELISA [36]. According to the instructions of ELISA Kit, the supernatant was collected by centrifugation homogenate (5,000 rpm for 15 min at 4°C). The activities of CXCL10, IL-12, IL-10, and CXCL2 were determined by ELISA Kit. Quantification of ELISA results was performed at 450 nm using an ELISA plate reader (Spectra Max M5, Molecular Devices, USA).

Colorectal
Cancer with Liver Metastasis Model. CRC with liver metastasis model was established via splenic injection model (SIM) [37] as described before. In brief, 50 athymic nude mice (BALA/c mice) were sterilized with 83% alcohol after inhalation anesthesia. A left lateral flank incision was made, and peritoneum was opened (at an approximate 8 mm length) to expose the spleen. CRC cells (2 × 10 6 / 100 μL) were injected slowly into the splenic parenchyma. After recovering from anesthesia, mice were returned to the cage. Fifteen days after SIM establishment, 200 μL exosomes were intraperitoneally injected. In vivo live imaging was conducted twice weekly to monitor metastatic spread. Twentyeight days after model establishment, mice were euthanized by dislocated cervical vertebrae, and liver tissue samples were collected and further examined by Western blot, IHC, qPCR, and immunofluorescence. All experimental procedures involved animals were conducted in accordance with the guidelines of the Animal Experimental Center of Zhejiang University School of Medicine and approved by the ethics committee of Zhejiang University School of Medicine (No. 22476); animals underwent all procedures under anesthesia, and every effort was made to minimize pain and death.
2.14. Immunofluorescence. Sections (5 μm) were stained by Immunofluorescence. Specifically, sections were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature and then blocked in 5% goat serum for 1 hr and labeled with primary antibodies against ZO1 (61-7300, Thermo Fisher, China, 1 : 100) and vimentin (5741 S, CST, China, 1 : 100) overnight at 4°C. After that, sections were incubated in secondary antibodies for 30 min at 37°C. Then, coverslips were sealed using Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). Images were acquired by laser scanning confocal microscopy (LSM710, Carl Zeiss, Germany) and analyzed using Image-Pro Plus 6.0.

Statistics Analysis.
Results were performed as mean value AE standard deviation (SD) and analyzed with Graph-Pad Prism (GraphPad Software, ver. 9.0.0, San Diego, CA). Statistical analyses were performed using one-way analysis of variance, followed by Turkey's post-test. Pearson's correlation analysis was performed to assess the relationship between CD66b and CD11b expression and clinicopathological status by SPSS statistical software (version 22.0, IBM, SPSS, USA). All experiments for cell culture were performed independently at least three times and in triplicate each time. Differences with p<0:05 were considered statistically significant.

Neutrophils Accumulation is Associated with Colorectal
Cancer Metastasis. To evaluate the distribution of neutrophils in TME, gene set enrichment analysis was performed via cancer immunome database (TCIA, https://tcia.at/home). The result revealed that neutrophils are relatively enriched in liver hepatocellular carcinoma, stomach adenocarcinoma, bladder cancer, lung adenocarcinoma, and CRC (Figure 1(a)). The fraction of immune cell profile in TME of CRC showed that neutrophils (32%), M1-like macrophage (27%), and M2-like macrophage (11%) are the most infiltrated immune cells in CRC (Figure 1(b)). Also, the results of immunohistochemistry showed the expression of neutrophils markers CD66b and CD11b located in stroma. Consistently, it is observed that the expression of CD66b and CD11b was increased in CRC tissues compared with para-cancerous tissues (Figure 1(c)-1(f)), which indicated that neutrophils were accumulated in CRC tissues. Also, as shown in Table 2, the CD11b expression is correlated with tumor site and the presence of invasion of serosa, while no correlation was found between CD66b and clinicopathologic parameters.

Exosome Derived from Neutrophils Aggravated
Aggressive Behavior of CRC. To further investigate the effect of exosome derived from neutrophils on CRC aggressive behavior, we first induced the differentiation of human myeloid leukemia cell line NB-4 cells by ATRA. Then, the polarization of neutrophil-like NB-4 cells to N2-like neutrophils was induced by TGF-β. CD11b is an important molecule for neutrophil chemotaxis [38]. In a normal situation, CD11b is mainly stored in intracytoplasmic granules and only expressed at low levels on the cell surface. After neutrophil activation, CD11b expression is upregulated, and it has been  found that CD11b can be a characteristic marker of neutrophil activation [39]. To determine the concentration of TGFβ, the proportion of CD11b and CD66b (biomarker of neutrophils) was detected via flow cytometer. As result shown, the proportion of CD11b and CD66b was increased with the increase of TGF-β concentration at 0.1 and 1 ng/mL. No statistical significance was observed at 1 and 10 ng/mL. The result indicated that, under the concentration of 1 and 10 ng/ mL, TGF-β activated the neutrophils the best (Figure 2(a)). Thereby, 10 ng/mL TGF-β was selected for the subsequence experiment. Interestingly, the proportion was markedly decreased at 20 ng/mL. Also, compared with non-treated neutrophils, the concentration of proinflammatory factors CXCL10 and IL-12 was observed no statistical significance in TGF-β-treated neutrophils, while after TGF-β treatment, the concentration of anti-inflammatory factors IL-10 and CCL2 was significantly increased, which is consistent with previous notes [25] (Figure 2(b)). Hence, TGF-β successfully induced the polarization of neutrophils into N2-like neutrophils. Then, the exosome was extracted from nontreated and N2-like neutrophils. The extracted exosomes were identified via TEM, and small membrane vesicles sized from 40 to 100 nm can be observed (Figure 2(c)). Also, the expression of exosome-related proteins CD63, CD81, TSG101 and Alix was increased in extracted exosomes compared to nontreated and N2-like neutrophils respectively ( Figure 2(d)).
Using PKH-67 to label exosomes, we observed that exosomes were taken by recipient COLO205 and SW480 ( Figure 3(a), Supplementary 2). After exosomes were taken up, the aggressive behavior of COLO205 and SW480 was investigated. CCK-8 was used to detect the proliferation of COLO205 and SW480 ( Figure 3(b), Supplementary 2). Compared with a control group, the proliferation of COLO205 and SW480 was increased significantly after cocultured with exosomes derived from N2-like neutrophils. Also, cell apoptosis was alleviated after the stimulation of exosomes derived from N2-like neutrophils (Figures 3(c) and 3(d), Supplementary 2). The results of Wound healing assay and Transwell assay were shown that the migration and invasion of COLO205 and SW480 were promoted by exosomes derived from N2-like neutrophils (Figure 3 .

miR-4780 Was Enriched in Exosome Derived from
Neutrophils and Aggravated EMT and Angiogenesis. Next, the exosomes derived from non-treated (N0-like) and N2-like neutrophils were collected, and microarray was used      Stem Cells International to find the differential expressed miRNAs in exosomes derived from N2-like neutrophils (Figure 4(a), Supplementary 1). According to our previous research [40], two miRNAs (select criteria: p<0:05, fold change >1.5, upregulated) was selected, i.e., hsa-miR-4780 and hsa-miR-513b-3p. Then qPCR was used to verify the expression of miRNAs (Figures 4(b) and 4(c)). The results showed both miRNAs were upregulated in N2-like neutrophils, which is coincident with the results from RNA sequencing. To find EMT-related miRNA, the target genes of hsa-miR-4780 and hsa-miR-513b-3p were predicted and then EMTrelated mRNAs were selected according to previous study [38]. We, therefore, chose hsa-miR-4780 for subsequent research. miR-4780 inhibitor and its corresponding negative control vector were transfected into N2-like neutrophils and the exosomes were extracted. CRC cell lines COLO205 and SW480 were cocultured with extracted exosomes. The intake of PKH-67 labeled exosomes was verified with immunofluorescence ( Figure 4(d), . Therefore, the overexpression of miR-4780 promoted aggressive behavior of COLO205 and SW480 and aggravated metastasis by increasing the incidence of EMT.
To detect the effect of miR-4780 on angiogenesis, we cocultured HUVECs with COLO205 and SW480, and purified neutrophils exosomes were then added. CCK-8 and Wound healing assay were performed to detect the proliferation and migration of HUVECs ( Figure 5(e)-5(g), Supplementary 2). After the intake of N2-like neutrophil exosome, the proliferation, and migration of HUVECS were increased compared to those with N0-like neutrophil exosomes. While, miR-4780 inhibitor retrieved the result. Also, the tube formation was conducted via BD Matrigel (Figures 5(h) and 5(i), Supplementary 2). Similarly, the tube formation was promoted by the intake of N2-like neutrophil exosomes compared with those cocultured with the N0-like neutrophil exosome. The result was retrieved by miR-4780 inhibitor. Also, same trend was observed in the expression of vascular endothelial growth factor (VEGF) and CD34 in HUVECs ( Figure 5(j)-5(l), Supplementary 2). Hence, N2-like neutrophil exosomal miR-4780 aggravated the angiogenesis by increasing the expression of VEGF and CD34 in HUVECs.

SOX11 Was a Target of miR-4780 and Aggravated EMT
and Angiogenesis. The expression of SOX11 was lower expressed in COAD from TCGA (Figure 6(a)). Also, lower expression of SOX11 indicated the favorable overall survival (OS) and disease-free survival (DFS) (Figures 6(b) and 6(c)). Then, the target of miR-4780 was predicted by Target Scan (Ver. 7.2, https://www.targetscan.org/vert_72/), and SOX11 was selected as a target of miR-4780 (Figure 6(d)). To further verify the relationship between miR-4780 and SOX11, dual fluorescence staining is performed (Figure 6(e)). In SOX11 wild-type group, when miR-4780 was overexpressed, relative luciferase activity was decreased. However, there was no significant difference between SOX11 mutate group. We can draw a conclusion that SOX11 was a target of miR-4780.
Next, to evaluate the function of SOX11 in CRC, pcDNA SOX11 was transfected into COLO205 and SW480, which absorbed the extracted exosomes. The expression of miR-4780 and SOX11 was detected via qPCR and western blot ( Figure 6(f )-6(h), Supplementary 2). The result showed that miR-4780 was highly expressed, while SOX11 was poorly expressed in N2-like neutrophils-derived exosomes. However, the transfection of pcDNA-SOX11 increased the expression. Also, the transfection of pcDNA-SOX11 suppressed the proliferation, migration, and invasion and aggravated the apoptosis of CRC cell lines compared with those only absorbed N2-like neutrophils (Supplementary 2). Further, the expression of Ecadherin was increased, and the expression of N-cadherin and vimentin was decreased after pcDNA-SOX11 transfection, which suggested the increase of SOX11 expression suppressed the incidence of EMT (Figure 6(i)-6(l), Supplementary 2).
When refers to the effect of SOX11on angiogenesis, the proliferation, migration, and tube formation ability of HUVECS were suppressed after pcDNA-SOX11 transfection compared with those only absorbed N2-like neutrophils ( Figure 6(p)-6(s), Supplementary 2). The expression of VEGF and CD34 (Figure 6(m)-6(o), Supplementary 2) also decreased after pcDNA-SOX11 transfection. Which suggested the increase of SOX11 expression suppressed the incidence of angiogenesis.

miR-4780 Can Aggravate EMT In Vivo.
Hepatic metastasis is prevalent in CRC; approximate 35%-55% of patients will develop hepatic metastasis. Thus, we cocultured COLO205 and SW480 with exosomes and injected the cells from teil veins, then investigated the hepatic metastasis in mice. The hepatic metastasis was monitored via in vivo live imaging. The hepatic metastasis was aggravated by N2-like neutrophil exosome, which was reversed by miR-4780 inhibitor. (Figure 7(a)) Then, the hepatic function was evaluated by the content of ALT, AST, GGT, and ALP. Obviously, the hepatic function was damaged by N2-like neutrophil exosome, which was reversed by miR-4780 inhibitor (Figure 7(b)). Also, the result of HE staining showed that a larger necrotic area was observed after N2-like neutrophil exosomes stimulation, while miR-4780 inhibitor reversed the result (Figure 7(c)). The expression of miR-4780 and SOX11 in liver tissue was detected. The expression of miR-4780 was increased after N2-like neutrophil Stem Cells International     (Figures 7(d) and 7(e)). Also, the expression of SOX11 was decreased after cocultured with N2-like neutrophil exosome, while the results was reversed by miR-4780 inhibitor. The expression of EMT-relative proteins in liver tissue was detected by western blot and immunofluorescence (Figures 7(f), 7(g), 7(i), and 7(j)). The results showed that the expression of epithelial surface makers (i.e., E-cadherin and occludin) was decreased by N2-like neutrophil exosome, while the expression of mesenchymal makers (i.e., N-cadherin, vimentin, and Zo1) was increased. Also, the expression of CD34 and VEGF was increased by N2-like neutrophil exosome (Figures 7(h), 7(k), and 7(l)). However, the deprive of miR-4780 reversed the expression, which indicated that miR-4780 derived from N2-like neutrophil exosome aggravated EMT and angiogenesis (Figure 8).

Discussion
In this study, we first verified the enrichment of neutrophils in tumor tissues harvested from CRC, and the expression of CD11b is correlated with tumor site and the presence of invasion of serosa. Also, we proved that the internalization of N2 exosomes aggravated the viability, migration, and invasion of CRC cell lines and suppressed the cell apoptosis.
To further investigate the molecular mechanism, we conducted the miRNA expression profile in the N2-like neutrophils and thereby choose hsa-miR-4780 for a subsequent experiment. We found that the overexpression of miR-4780 from N2-like neutrophil-derived exosome aggravated the EMT and angiogenesis. Moreover, miR-4780 can regulate its target gene SOX11 to affect the EMT and angiogenesis of CRC cell lines. CRC with liver metastasis model also verified that abnormal expression of miR-4780 from N2-like neutrophil-derived exosome aggravated the metastasis and development of tumor via EMT and angiogenesis. The presence of neutrophils in tumors has traditionally been regarded as indicative of a failed immune response against cancer [41]. However, recent reports have revealed multiple roles for neutrophils besides immune response signaling. In this manuscript, the analyzed result from TCIA showed that neutrophils are relatively enriched in CRC and is one of the most infiltrated immune cells (approximately 32%). Also, neutrophils were enriched in the tumor tissues we harvested from primary CRC patients. The relationship between TAN and human cancer has not been systemically investigated, although the increase of neutrophils counts in peripheral blood, also known as neutrophils-to-lymphocyte ratio (NLR), has been regarded as a poor prognostic factor in CRC [42]. Ashizawa et al. [43] showed that decreasing post-NLR was related to a better OS rate and DFS even in high pre-NLR patients with CRC. Li et al. [44] retrospectively analyzed a cohort of 354 CRC patients and revealed that the NLR was an independent prognostic factor for OS in early-stage colon cancer. Ye et al. [33] also proved that the number of CD66b + TANs, Tregs, and TAMs were more reliable than traditional indicators for evaluating prognosis in CRC patients. In this manuscript, we found that the expression of CD11b is correlated with tumor site and the           presence of invasion of serosa. Many lineages of stromal cells contained in TEM have been reported to promote the growth of CRCs, including TAN. Intriguingly, the role of neutrophils in tumor growth and metastasis was controversial. This can be explained by the phenotypically plastic of neutrophils with similarities to corresponding macrophage populations [27]. Accumulated results suggested that tumors can induce a pro-tumor phenotype in neutrophils that, in turn, help tumor progression. Raccosta et al. [45] have shown that oxysterol derived from tumor cells recruit protumor neutrophils, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression. Fridlender et al. [25] showed that TGF-β within the TME induces the phenotypically plastic of neutrophils and increased the activation and population of TAN with a protumor phenotype. Neutrophil elastase and prostaglandin E2 secreted by neutrophils are proved to promote tumor cell proliferation [46,47]. In line with these results, we found that exosomes derived from TGF-β induced N2-like neutrophils can also promote the growth of CRC. Metastasis is the primary contributor to death in patients with solid cancers. Largely due to our poor understanding on the cellular and molecular process associated with metastasis, once the tumor has spread from the primary site, neither conventional chemotherapy nor current targeted therapy offers significant benefits [48]. Despite that CRC is the result of multiple factors, metastasis is an important factor that affects the survival and prognosis of CRC. At least one-third of CRC patients develop liver metastases [49]. Reports show that the 5-year survival rate of patients with early CRC can reach more than 90%, while the rate drops to 69.2% for patients with local metastasis and less than 10% for patients with distant metastasis [50]. EMT is a cellular process in which cell lost their epithelial characteristics and acquire mesenchymal features [51]. EMT has been reported to be involved in various tumor functions, including tumor initiation, malignant progression, tumor stemness, tumor cell migration, intravasation to the blood, metastasis, and resistance to therapy. Li et al. [52] proved that TANs produce IL-17a and promote EMT of GC cells via JAK2/STAT3 signaling. Likewise, Zhang et al. [24] proved that interaction with neutrophils aggravated the migration and invasion through the activation of the ERK pathway and the induction of EMT in gastric cancer. Angiogenesis is a critical step not only in tumor development but in distant metastases. Accumulated results suggested that except tumor cells, stromal cells in the TME also participated in the process of angiogenesis. Neutrophils recruited to malignant site produces matrix metalloproteinase-9 and VEGF and therefore participates in the incidence of angiogenesis [53]. Shojaei et al. [54] showed that BV-8 overexpression modulates the mobilization of CD11b + Gr1 + neutrophils and therefore promotes angiogenesis. Consistent with these results, we found hsa-miR-4780 in exosome derived by N2-like neutrophils promote EMT and angiogenesis in CRC.
SOX11 is a member of sex-determining region Y-related a high mobility group-box (SOX) family, which are characterized as transcription factors with a high mobility group (HMG) DNA-binding domain [55]. Based on the protein sequence alignment, the SOX gene family was divided into 10 groups from A to J. SOX11, together with SOX4 and SOX12 are members of subgroup C, which bind to the 5′-(A/T) (A/T) CAA(A/T) G-3′ consensus site, and thus induce several DNA conformational changes that allow other transcription factors/regulatory proteins to bind together [56]. Similar to SOX4, SOX11 has been reported to be essential for organogenesis, neurodevelopment, and neuronal growth. Recently, SOX11 was reported as a key oncogenic factor in mantle cell lymphoma. Moreover, SOX11 and its hypermethylation were reported to be involved in the progression of head and neck cancer [57], cervical cancer [58], prostate cancer [59], etc. Wang et al. [60] proved that miR-31 modulates EMT in papillary thyroid carcinoma by targeting SOX11. Likewise, SOX11 was reported to promote angiogenesis in mantle cell lymphoma [61,62]. However, other reports have shown that SOX11 plays an oncogenic role in : miR-4780 derived from N2-like neutrophil exosome aggravated EMT and angiogenesis. Exosome derived from neutrophils was extracted, and differentially expressed miRNA in neutrophils-derived exosomes has been sequenced by microarray profile. In this manuscript, we found that hsa-miR-4780 in exosome derived by N2-like neutrophils promotes EMT and angiogenesis by targeting SOX11 in the colorectal cancer. 22 Stem Cells International small-cell lung cancer [63] and acute myeloid leukemia [64]. This suggests that the differential role of SOX11 in tumors may be related to tissue specificity and TME. In this manuscript, we found that hsa-miR-4780 in exosome derived by N2-like neutrophils promotes EMT and angiogenesis by targeting SOX11 in CRC. However, there are also some limitations existed in this manuscript. The limited number of patient samples due to difficulties in tissue collection, especially in tumor tissue from patients with distant metastases, may account for the lack of correlation found between CD66b and clinicopathological parameters. Circulating tumor cells (CTCs), which are shed from primary or secondary tumors and actively invade the bloodstream, are regarded as one of the predominant factors for distant metastases [65,66]. Wei et al. [67] indicated that tumor-associated macrophages-induced EMT programs can enhance the migration and invasion of CTCs, and in turn promote macrophage recruitment. Whether similar crosstalk can be validated between CTCs and N2-like TAN deserves further investigation.
In conclusion, for the first time, our current finding discloses an important mechanism that mR-4780 from N2-like neutrophil-derived exosome aggravated the metastasis and development of tumor via EMT and angiogenesis.

Data Availability
All data, models, or code generated or used during the study are available from the corresponding author by request.